U.S. patent number 10,655,158 [Application Number 15/572,555] was granted by the patent office on 2020-05-19 for bacterial counting method.
This patent grant is currently assigned to Jiangsu University. The grantee listed for this patent is Jiangsu University. Invention is credited to Xuetao Hu, Xiaowei Huang, Zhihua Li, Jiyong Shi, Xucheng Zhou, Xiaobo Zou.
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United States Patent |
10,655,158 |
Zou , et al. |
May 19, 2020 |
Bacterial counting method
Abstract
Disclosed is a bacterial counting method, comprising the
following steps, S1: preparing a polyaniline/bacterial composite
film on the surface of a glassy carbon electrode by electric
polymerization; S2: drawing the standard curve of the
polyaniline/bacterial composite film modified electrode; and S3:
determining the bacterial concentration of the bacteria solution
sample to be tested according to the standard curve obtained in
step S2. The method does not require the cultivation of the
bacteria in the implementation process, such that same takes a
short time and is easy to operate; the method uses aniline as the
main reagent, and the consumption of the solution to be tested is
small, such that the testing cost is low and the equipment is
simple; the method has the advantages of a good repeatability and a
wide detection linear range; and the method is an easy to operate,
short time-consuming, and low cost bacterial counting method. The
method is a bacterial counting method based on the
polyaniline/bacterial composite film, and can achieve rapid and
accurate detection of the bacterial thallus concentration.
Inventors: |
Zou; Xiaobo (Jiangsu,
CN), Li; Zhihua (Jiangsu, CN), Shi;
Jiyong (Jiangsu, CN), Huang; Xiaowei (Jiangsu,
CN), Zhou; Xucheng (Jiangsu, CN), Hu;
Xuetao (Jiangsu, CN) |
Applicant: |
Name |
City |
State |
Country |
Type |
Jiangsu University |
Zhenjiang, Jiangsu |
N/A |
CN |
|
|
Assignee: |
Jiangsu University (Zhenjiang,
Jiangsu, CN)
|
Family
ID: |
55555627 |
Appl.
No.: |
15/572,555 |
Filed: |
March 30, 2016 |
PCT
Filed: |
March 30, 2016 |
PCT No.: |
PCT/CN2016/077794 |
371(c)(1),(2),(4) Date: |
November 08, 2017 |
PCT
Pub. No.: |
WO2017/107333 |
PCT
Pub. Date: |
June 29, 2017 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20180155755 A1 |
Jun 7, 2018 |
|
Foreign Application Priority Data
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|
|
|
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Dec 21, 2015 [CN] |
|
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2015 1 0971248 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M
1/34 (20130101); G01N 15/0606 (20130101); G01N
15/1031 (20130101); G01N 15/0656 (20130101); C12Q
1/06 (20130101); G01N 2015/0065 (20130101); G01N
2015/1062 (20130101) |
Current International
Class: |
C12Q
1/06 (20060101); C12M 1/34 (20060101); G01N
15/06 (20060101); G01N 15/10 (20060101); G01N
15/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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101003830 |
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Jul 2007 |
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CN |
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101216449 |
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Jul 2008 |
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CN |
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101216449 |
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Jul 2008 |
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CN |
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104198558 |
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Dec 2014 |
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CN |
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104407031 |
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Mar 2015 |
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CN |
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105067694 |
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Nov 2015 |
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CN |
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Other References
Sivaprasad et al. Glassy Carbon Electrode Modified With Polyaniline
Based Nanosensors for Electrochemical Determination of Aurone
Flavonoid; International Journl of PharmaSciences and Research,
vol. 6, No. 1, pp. 129- (Year: 2015). cited by examiner .
Zhihua et al. Bacteria Counting Method Based on
Polyaniline/Bacteria Thin Film; Biosensors and Bioelectronics, vol.
81, pp. 75-79. (Year: 2016). cited by examiner .
International Search Report for PCT/CN2016/077794, dated Jul. 26,
2016. cited by applicant .
Gao et al., "On bacteria biosensor based on E. coli modified carbon
paste electrode by cyclic voltammetry," Journal of Shenyang Normal
University (Natural Science Edition) 29(2):305-308 (2011). cited by
applicant.
|
Primary Examiner: Switzer; Juliet C
Assistant Examiner: Martin; Paul C
Attorney, Agent or Firm: Medler Ferro Woodhouse Mills
PLLC
Claims
The invention claimed is:
1. A bacteria counting method, comprising the following steps: (a)
preparing a polyaniline/bacterial composite film on a surface of a
glassy carbon electrode via an electro-polymerization method,
comprising: 1) preparing a standard bacterial suspension,
comprising: preparing a bacterial culture solution; carrying out
autoclaved sterilization of the bacterial culture solution;
inoculating a proper amount of strains for culturing; and
centrifuging and washing the bacteria solution obtained after
culturing to obtain the standard bacterial suspension; 2) fixing of
bacteria on the surface of the glassy carbon electrode, comprising:
pretreating the glassy carbon electrode and measuring a cyclic
voltammetry curve of the glassy carbon electrode, until a potential
difference of the oxidation-reduction peak is reduced to be within
80 mV; drying the glassy carbon electrode; dripping the standard
bacterial suspension to the surface of the glassy carbon electrode;
and drying, thereby fixing bacteria to the surface of the glassy
carbon electrode; and 3) polymerization of phenylamine on the
electrode surface, comprising: putting the glassy carbon electrode
fixed with the bacteria into a sulfuric acid solution containing
phenylamine; scanning via cyclic voltammetry; and making
phenylamine polymerize on the surface of the glassy carbon
electrode to obtain the polyaniline/bacterial composite film; (b)
drawing a standard curve of a modified electrode of the
polyaniline/bacterial composite film, comprising: 1) carrying out
gradient dilution of the standard bacterial suspension obtained in
step (a) sequentially to acquire bacterial suspensions with
different concentrations; 2) preparing a polyaniline/bacterial
composite film on the surface of the glassy carbon electrode for
each concentration of the bacterial suspensions with different
concentrations according to step (a) and measuring a cyclic
voltammetry curve for each concentration of bacterial suspension;
and 3) drawing a standard curve of the modified electrode of the
polyaniline/bacterial composite film by taking the logarithm of
bacterial concentration as horizontal coordinates and taking peak
current corresponding to the peak of the cyclic voltammetry curve
obtained in step (b)(2) as vertical coordinates; and (c) measuring
a bacterial concentration of a bacteria solution sample according
to the standard curve obtained in the step (b).
2. The bacteria counting method according to claim 1, wherein the
sulfuric acid solution is 0.5M; phenylamine is 0.1M; scanning is
carried out with 1 to 20 cycles at a scanning rate of 5 to 100 mV/s
in the cyclic voltammetry; a lower limit of scanning voltage is
-0.6V to 0V; and an upper limit of scanning voltage is 0.75V to
1.2V.
3. The bacteria counting method according to claim 2, wherein
scanning is carried out with 10 cycles; the scanning rate is 50
mV/s; and the scanning voltage ranges from -0.2V to 0.9V.
4. The bacteria counting method according to claim 1, wherein step
(b)(2) comprises: preparing the polyaniline/bacterial composite
film on the surface of the glassy carbon electrode for each
concentration of the bacterial suspensions with different
concentrations according to step (a); rinsing the
polyaniline/bacterial composite film with distilled water; and
measuring the cyclic voltammetry curve for each concentration of
bacterial suspension in 0.1 M of H.sub.2SO.sub.4 solution at
scanning rate of 50 mV/s and under scanning voltage ranging from
-0.2 to 0.9V.
5. The bacteria counting method according to claim 1, wherein step
(c) comprises: 1) preparing a further polyaniline/bacterial
composite film on the surface of the glassy carbon electrode by
utilizing the bacterial solution sample to be measured in
accordance with step (a); and measuring a cyclic voltammetry curve
of the bacterial solution sample to be measured; and 2) determining
peak current of the cyclic voltammetry curve of the bacterial
solution sample to be measured, and calculating bacterial
concentration in accordance with the standard curve of the modified
electrode of the polyaniline/bacterial composite film obtained in
the step (b).
Description
FIELD OF THE INVENTION
The present invention belongs to the fields of material science and
microbiology, and particularly relates to a bacteria counting
method based on a polyaniline/bacterial composite film.
BACKGROUND OF THE INVENTION
It will be of great importance for the detection of the cell
concentration of bacteria in the fields of microbial fermentation,
environmental monitoring, foods quality inspection, etc. Currently,
the common bacteria counting method is culture method, including a
plate counting method and a MPN method.
The plate counting method is a national standard inspection method
for detecting the total number of bacterial colonies (see
reference: GB/T 4789.2-2010 Microbiological examination of food
hygiene--Detection of aerobic bacterial count [S]), which is a
standard method for judging the accuracy of other counting methods.
The method is accurate in single colony counting in a dish,
however, as the bacterial colonies in the culture dish are uniform
in density, it will be subjective and inaccurate in the artificial
observation and counting of adhesion colonies. As a whole, the
method is complex in operation, long in time consumption, large in
error and low in efficiency.
MPN is a diluent culture counting method. The method is suitable
for detecting foods with a large number of competitive bacteria and
raw materials thereof and untreated food containing a little amount
of Staphylococcus aureus. The MPN method is a common indirect
counting method. However, there is a certain limitation for such
method.
Such culture methods have the defects of long time consumption,
strict sterile operation, complex procedures, high labor and large
influence by the culture condition.
In order to overcome the defects of the culture method, a plurality
of counting methods are widely used. Among these methods, the
fluorescence microscope counting method and the flow cytometry
counting method are reliable and widely accepted. For the
fluorescence microscope counting method, the fluorescence
microscope use ultraviolet rays as a light source for irradiating
detected objects to make them emit fluorescent light, and then, the
shapes, positions and quantity of the objects are observed under
the microscope. In the flow cytometry counting method, particles
passing through the laser beam are counted via laser. When
particles or cells pass through the laser beam, there will be
refraction and reflection on light rays. The refraction signal and
the reflection signal are recorded by a detector. A peak will be
generated as soon as a cell passes through the laser beam, and
finally, the number of peaks is recorded.
Although such two methods have the characteristics of quickness and
accuracy, equipment is expensive and the cost of using and
maintenance is high. Hence, it will have the important value to
seek a bacteria counting method with simple and quick operation and
low cost.
SUMMARY OF THE INVENTION
In order to solve the defects in the prior art, the present
invention, provides a bacteria counting method with simple
operation, short time consumption and low cost, which can realize
the quick and accurate detection of the cell concentration of
bacteria.
The technical solution of the present invention is: a bacteria
counting method, comprising the following steps:
S1: preparing a polyaniline/bacterial composite film on the surface
of a glassy carbon electrode via the electro-polymerization
method;
S2: drawing a standard curve of a modified electrode of the
polyaniline/bacterial composite film; and
S3: measuring the bacterial concentration of a bacteria solution
sample to be measured according to the standard curve obtained in
the step S2.
In the above-mentioned solution, the step S1 specially comprises
the following steps: 1) preparation of standard bacterial
suspension: preparing bacterial culture solution, carrying out
autoclaved sterilization, inoculating proper amount of strains for
culturing, and centrifuging and washing bacteria solution obtained
after culturing to obtain the standard bacterial suspension; 2)
fixing of bacteria on the surface of the glassy carbon electrode:
pretreating the glassy carbon electrode, measuring its cyclic
voltammetry curve until the potential difference of the
oxidation-reduction peak is reduced to be within 80 mV, drying the
glassy carbon electrode, dripping the standard bacterial suspension
to the surface of the glassy carbon electrode, drying, and fixing
bacteria to the surface of the glassy carbon electrode; and 3)
polymerization of phenylamine on the electrode surface: putting the
glassy carbon electrode fixing with the bacteria into sulfuric acid
solution containing phenylamine, scanning via cyclic voltammetry,
and making phenylamine polymerize on the surface of the glassy
carbon electrode to obtain the polyaniline/bacterial composite
film.
In the above-mentioned solution, the sulfuric acid solution is
0.5M, phenylamine is 0.1M, scanning is carried out with 1 to 20
cycles at a scanning rate of 5 to 100 mV/s in the cyclic
voltammetry, the lower limit of voltage is -0.6 to 0V, and the
upper limit of voltage is 0.75 to 1.2 V.
Preferably, the scanning is carried out with 10 cycles, the
scanning rate is 50 mV/s, and the scanning voltage ranges from -0.2
to 0.9 V.
In the above-mentioned solution, the step S2 specially comprises
the following steps:
1) carrying out gradient dilution of the standard bacterial
suspension obtained in the step S1 sequentially to acquire
bacterial suspensions with different concentrations;
2) preparing the polyaniline/bacterial composite film on the
surface of the glassy carbon electrode respectively by utilizing
the obtained bacterial suspensions with different concentrations in
accordance with the step S1, and rinsing with distilled water,
measuring its cyclic voltammetry curve in 0.1 M of H.sub.2SO.sub.4
solution at a scanning rate of 50 mV/s and under scanning voltage
ranging from -0.2 to 0.9 V; and
3) drawing a standard curve of the modified electrode of the
polyaniline/bacterial composite film by taking the logarithm of
bacterial concentration as horizontal coordinates and taking peak
current corresponding to the peak of the cyclic voltammetry curve
obtained in 2) as vertical coordinates.
In the above-mentioned solution, the step S3 specially comprises
the following steps:
1) preparing the polyaniline/bacterial composite film on the
surface of the glassy carbon electrode by utilizing the bacterial
suspension to be measured in accordance with the step S1, and
measuring its cyclic voltammetry curve in accordance with the step
S2; and
2) determining the peak current of the cyclic voltammetry curve,
and calculating bacterial concentration in accordance with the
standard curve of the modified electrode of the
polyaniline/bacterial composite film obtained in the step S2.
Compared with the prior art, the present invention has the
following advantages:
1. The polyaniline/bacterial composite film is prepared on the
surface of the glassy carbon electrode via the
electro-polymerization method. The bacteria fixed to the surface of
the glassy carbon electrode can hinder the polymerization of the
phenylamine on the electrode surface, so that the prepared
polyaniline/bacterial composite film has different electrochemical
properties, which can realize the quick and accurate detection of
the cell concentration of bacteria.
2. In the implementation process of the present invention, it is
not necessary for the bacteria to be counted after being cultured
like the conventional plate counting method, hence, the method of
the present invention is short in time consumption and easy to
operate.
3. In the present invention, phenylamine is used as a main reagent,
and the consumption of the solution to be measured is little,
hence, the detection costs are low, and equipment is simple.
4. The method of the present invention has the advantages of good
repeatability and wide linearity range of detection for the
measurement of the cell concentration of the same bacteria.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is the schematic diagram of the integral preparation process
of the polyaniline/bacterial composite film.
FIG. 2(a) is the change diagram of the cyclic voltammetry curve of
the electrode in the process of preparing the polyaniline-bacterial
composite film via the cyclic voltammetry.
FIG. 2(b) is the morphology comparison diagram of the prepared
polyaniline/bacterial composite film and the pure polyaniline
film.
FIG. 3(a) is the cyclic voltammetry curve graph of the modified
glassy carbon electrode of the polyaniline/bacterial composite film
in 0.1M of H.sub.2SO.sub.4 solution prepared by using the bacterial
suspension of Bacillus subtilis with different concentrations.
FIG. 3(b) is the standard curve graph of the cyclic voltammetry
curve of the modified electrode of the polyaniline/Bacillus
subtilis film at 0.2 V peak current.
FIG. 4 is the detection result comparison diagram of the bacterial
suspension samples of Bacillus subtilis with different
concentrations, under the plate colony counting method and the
polyaniline/bacterial film counting method of the present
invention.
FIG. 5 is the standard curve graph of the cyclic voltammetry curve
of the modified electrode of the polyaniline/Escherichia coli film
at 0.2 V peak current.
FIG. 6 is the standard curve graph of the cyclic voltammetry curve
of the modified electrode of the polyaniline/Streptococcus
thermophilus film at 0.2 V peak current.
DETAILED DESCRIPTION OF THE INVENTION
Hereunder the present invention will be further detailed in the
specific embodiments of Bacillus subtilis, Escherichia coli and
Streptococcus thermophilus with reference to the accompanying
drawings, however, the protecting scope of the present invention is
not limited to the embodiments.
Example 1
Phenylamine, sulfuric acid, yeast extract and tryptone used in the
present invention are purchased from Sinopharm Group Chemical
Reagent Co., Ltd., and phenylamine is used after being distilled
under reduced pressure. In electrochemical measurement, the CHI660D
electrochemical workstation is adopted (Shanghai Chenhua Instrument
Co., Ltd.). In the present example, it will make description by
taking Bacillus subtilis as an example, and strains are purchased
from China Center of Industrial Culture Collection.
The bacteria counting method comprises the following steps:
S1. Preparing a polyaniline/bacterial composite film on the surface
of a glassy carbon electrode via the electro-polymerization
method;
FIG. 1 shows the integral preparation process of the
polyaniline/bacterial composite film, including the following
steps: (1) preparation of standard bacterial suspension: taking 5 g
of yeast extract, 10 g of tryptone and 10 g of sodium chloride,
adding water to prepare 1 L of culture solution, regulating pH to
7.0 by using NaOH, carrying out autoclaved sterilization at
temperature of 121.degree. C. for 20 min, inoculating proper amount
of Bacillus subtilis strains, and culturing in a constant
temperature incubator at temperature of 37.degree. C. for 20 h;
centrifuging the obtained bacteria solution under 3500 g of
centrifugal force at temperature of 4.degree. C. for 10 min and
then washing for 3 times to obtain standard Bacillus subtilis
suspension, and measuring its cell concentration via the plate
colony counting method, which is 5.33.times.108 CFUmL.sup.-1; (2)
fixing of bacteria on the surface of the glassy carbon electrode:
polishing the glassy carbon electrode sequentially by using
abrasive paper for metallograph, 0.3 .mu.m and 0.05 .mu.m of Al2O3
powder, carrying out ultrasonic washing respectively by using
ethanol and water, measuring the cyclic voltammetry (CV) curve of
the glassy carbon electrode in 1 mM of K.sub.3[Fe(CN).sub.6]
solution until the potential difference of the oxidation-reduction
peak is reduced to be within 80 mV, and drying to ensure the
consistent initial state of the electrode; dripping 10 .mu.L of
standard Bacillus subtilis suspension to the surface of the glassy
carbon electrode, and drying in a drying oven at temperature of
50.degree. C. for 15 min to fix bacteria to the surface of the
glassy carbon electrode, and obtaining the bacteria/glassy carbon
electrode; and (3) polymerization of phenylamine on the electrode:
putting the glassy carbon electrode fixing with bacteria into
sulfuric acid solution (0.5M) containing 0.1 M of phenylamine, and
scanning by taking the platinum filament electrode as the counter
electrode and taking the silver/silver chloride electrode as the
reference electrode via the cyclic voltammetry. In order to acquire
higher detection sensitivity, preferably, the number of scanning
cycles is 10, the scanning rate is 50 mV/s, the lower limit of
voltage is -0.2 V, the upper limit of voltage is 0.9 V, and the
polyaniline/Bacillus subtilis composite film is obtained. FIG. 2(a)
is the change of the cyclic voltammetry curve of the electrode in
the process of preparing the polyaniline/bacterial composite film
via the cyclic voltammetry, in which there are 3 pairs of typical
oxidation-reduction peaks: I a/I c, IIa/IIc, and IIIa/IIIc,
indicating the successful polymerization of phenylamine on the
electrode surface. In addition, the scanning current is gradually
increased with the increase of the scanning cycles, which manifests
the self-catalysis effect of polyaniline during the polymerization
of polyaniline. FIG. 2(b) is the morphology comparison of the
polyaniline/Bacillus subtilis composite film prepared at bacterial
concentration of 5.33.times.105 CFUmL-1 and the pure polyaniline
film, two films are apparently different in morphology, indicating
the fixing of stains possesses a large impact on the deposition of
polyaniline on the electrode surface, which shows different
electrochemical properties, and thereby providing basis for the
quantitative determination of cell concentration.
S2. Drawing a standard curve of a modified electrode of the
polyaniline/bacterial composite film:
Diluting the standard bacterial suspension with the concentration
of 5.33.times.108 CFUmL-1 obtained in the step S1 to be
1.066.times.108 CFUmL-1 , 5.33.times.107 CFUmL-1, 5.33.times.106
CFUmL-1, 1.066.times.106 CFUmL-1, 5.33.times.105 CFUmL-1 and
5.33.times.104 CFUmL-1 sequentially, preparing the
polyaniline/Bacillus subtilis composite film on the surface of the
glassy carbon electrode by utilizing the obtained bacterial
suspensions with different concentrations in accordance with the
step S1, rinsing by using distilled water, and measuring its cyclic
voltammetry curve in 0.1M of H2 SO4 solution. The scanning voltage
ranges from -0.2 to 0.9 V, and scanning rate is 50 mV/s.
Drawing a standard curve of the modified electrode of the
polyaniline/Bacillus subtilis composite film by taking the
logarithm of bacteria concentration as horizontal coordinates X and
taking peak current of the cyclic voltammetry curve at 0.2V as
vertical coordinates Y. FIG. 3(a) is the cyclic voltammetry curve
corresponding to the bacterial suspensions with different
concentrations, and FIG. 3(b) is its peak current standard curve.
As can be seen from FIG. 3, there is good linear relation between
the peak current of the electrode at 0.2V and the logarithm of
bacteria concentration: Y=-30.413X+272.560, and R.sup.2=0.982.
S3. Measuring the bacterial concentration of a bacteria solution
sample to be measured:
Preparing the polyaniline/Bacillus subtilis composite film by
utilizing the bacterial suspension in accordance with the step S1,
measuring the cyclic voltammetry curve of the film in 0.1 M of
H.sub.2SO.sub.4 solution in accordance with the step S2, and
calculating its cell concentration via the standard curve of the
modified electrode of the polyaniline/Bacillus subtilis composite
film obtained in the step S2 in accordance with the peak current of
it at 0.2V. Table 1 and FIG. 4 are the measuring result comparison
of the polyaniline/bacterial composite film counting method and the
plate colony counting method. The result indicates that the mean
value of the bacterial suspension concentration measured in the
method of the present invention for 5 times is basically consistent
with the measuring result of the plate colony counting method, but
the relative standard deviation of the method is obviously inferior
to that of the plate colony counting method, indicating that it has
the higher stability. The polyaniline/bacterial composite film
counting method is superior to the conventional plate colony
counting method in the counting result.
TABLE-US-00001 TABLE 1 Result comparison of the Bacillus subtilis
sample measured based on the polyaniline/bacterial composite film
counting method and the plate colony counting method Items
Polyaniline/bacterial composite Plate colony counting method film
counting method Mean value Relative standard Mean value Relative
standard Number (CFU mL.sup.-1) deviation (CFU mL.sup.-1) deviation
Sample 1 9.72 .times. 10.sup.4 9.57% 9.21 .times. 10.sup.4 5.21%
Sample 2 4.37 .times. 10.sup.5 15.29% 4.59 .times. 10.sup.5 5.49%
Sample 3 7.54 .times. 10.sup.7 8.73% 8.11 .times. 10.sup.7
3.87%
Example 2
The method used for this example is the same as that of example 1,
but the difference is that in this example, it will make
description by taking Escherichia coli as an example, and strains
are purchased from China Center of Industrial Culture
Collection.
The standard Escherichia coli suspension with cell concentration of
9.26.times.108 CFUmL.sup.-1 is obtained in the same method as the
step S1 in the example 1, and then is sequentially diluted to be
9.26.times.107 CFUmL.sup.-1, 9.26.times.106 CFUmL.sup.-1,
9.26.times.105 CFUmL.sup.-1, 9.26.times.104 CFUmL.sup.-1 and
9.26.times.103 CFUmL.sup.-1.
Drawing a standard curve of the modified electrode of the
polyaniline/Escherichia coli composite film by taking the logarithm
of Escherichia coli concentration as horizontal coordinates X and
taking peak current of the cyclic voltammetry curve at 0.2V as
vertical coordinates Y in accordance with the step S2 in example 1.
As can be seen in FIG. 5, there is good linear relation between the
peak current of the electrode at 0.2 V and the logarithm of
Escherichia coli concentration: Y=-24.249 X+217.33, and
R.sup.2=0.996.
Measuring the sample of the Escherichia coli bacteria solution to
be measured in accordance with step S3 in example 1. Table 2 is the
result comparison of the Escherichia coli sample measured based on
the polyaniline/bacterial composite film counting method and the
plate colony counting method. The result indicates that the mean
value of the bacterial suspension concentration measured in the
method of the present invention for 5 times is basically consistent
with the measuring result of the plate colony counting method, but
the relative standard deviation of the method is obviously inferior
to that of the plate colony counting method, indicating that it has
the higher stability. The polyaniline/bacterial composite film
counting method is also applied to the Escherichia coli sample.
TABLE-US-00002 TABLE 2 Result comparison of the Escherichia coli
sample measured based on the polyaniline/bacterial composite film
counting method and the plate colony counting method Items
Polyaniline/bacterial composite Plate colony counting method film
counting method Mean value Relative standard Mean value Relative
standard Number (CFU mL.sup.-1) deviation (CFU mL.sup.-1) deviation
Escherichia Sample 1 8.12 .times. 10.sup.4 8.66% 8.72 .times.
10.sup.4 4.31% coli Sample 2 5.23 .times. 10.sup.6 15.29% 4.97
.times. 10.sup.6 5.19%
Example 3
The method used for this example is the same as that of example 1
and example 2, but the difference is that in this example, it will
make description by taking Streptococcus thermophilus as an
example, and strains are purchased from China Center of Industrial
Culture Collection.
Weighing 52.4 g of MRS bouillon culture-medium (purchased from
Qingdao Haibo Biotechnology Co., Ltd.), heating to dissolve in 1 L
of distilled water, carrying out autoclaved sterilization at
temperature of 118.degree. C. for 15 min, cooling, inoculating
proper amount of Streptococcus thermophilus strains, and culturing
in the constant-temperature incubator at temperature of 37.degree.
C. for 20 h. Centrifuging the obtained bacteria solution at
temperature of 4.degree. C. under 3500 g of centrifugal force for
10 min, and then washing for 3 times, obtaining the standard
Streptococcus thermophilus suspension, measuring its cell
concentration via the plate colony counting method, which is
6.72.times.10.sup.9 CFUmL.sup.-1. Sequentially diluting the
standard suspension to be 6.72.times.10.sup.8 CFUmL.sup.-1,
6.72.times.10.sup.7 CFUmL.sup.-1, 6.72.times.10.sup.6 CFUmL.sup.-1,
6.72.times.10.sup.5 CFUmL.sup.-1 and 6.72.times.10.sup.4
CFUmL.sup.-1. Drawing a standard curve of the modified electrode of
the polyaniline/Streptococcus thermophilus composite film by taking
the logarithm of bacterial concentration as horizontal coordinates
X and taking peak current of the cyclic voltammetry curve at 0.2V
as vertical coordinates Y in accordance with the step S2 in the
example 1. As can be seen from FIG. 6, there is good linear
relation between the peak current of the electrode at 0.2V and the
logarithm of bacterial concentration: Y=-28.601 X+278.430, and
R.sup.2=0.988.
Measuring the sample of the Streptococcus thermophilus bacteria
solution to be measured in accordance with step S3 in example 1.
Table 3 is the result comparison of the Streptococcus thermophilus
sample measured based on the polyaniline/bacterial composite film
counting method and the plate colony counting method. The result
indicates that the mean value of the bacterial suspension
concentration measured in the method of the present invention for 5
times is basically consistent with the measuring result of the
plate colony counting method, but the relative standard deviation
of the method is obviously inferior to that of the plate colony
counting method, indicating that it has the higher stability. The
polyaniline/bacterial composite film counting method is also
applied to the Streptococcus thermophilus sample.
TABLE-US-00003 TABLE 3 Result comparison of the Streptococcus
thermophilus sample measured based on the polyaniline/bacterial
composite film counting method and the plate colony counting method
Items Polyaniline/bacterial composite Plate colony counting method
film counting method Mean value Relative standard Mean value
Relative standard Number (CFU mL.sup.-1) deviation (CFU mL.sup.-1)
deviation Streptococcus Sample 1 3.22 .times. 10.sup.8 10.54% 3.61
.times. 10.sup.8 4.81% thermophilus Sample 2 2.89 .times. 10.sup.5
11.76% 3.13 .times. 10.sup.5 5.24%
The above mentioned examples are the preferable embodiments of the
present invention; however, the present invention is not limited to
the above-mentioned embodiments. The person skilled in the art
could make apparent improvement, replacement or change under the
condition of not deviating from the essential content of the
present invention, which belongs to the protection scope of the
present invention.
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